Signal transduction protein involved in plant dehiscence

ABSTRACT

This invention relates to novel plant nucleic acid sequences and proteins. The sequences and proteins are useful in the control of plant dehiscence and in the production of male sterile plants. According to a first aspect of the invention there is provided nucleic acid optionally encoding a signal transduction protein involved in the process of dehiscence. Such a sequence or signal transduction protein has never previously been described in plant dehiscence.

[0001] This invention relates to novel plant nucleic acid sequences andproteins. The sequences and proteins are useful in the control of plantdehiscence and in the production of male sterile plants.

[0002] The production of seed is an important developmental process inall higher plants. In oilseed rape (Brassica napus), followingabscission of floral parts, pods or siliques are formed which contain15-30 seeds. Around 50-70 days after anthesis (DAA) the pods becomesusceptible to shatter, a process that serves to expel the mature seedsinto the surrounding environment. In the days leading to dehiscence, anarray of anatomical, molecular and biochemical changes take place, thuspreparing both seed and pod for the event. Shatter eventually occurs asa result of a combination of factors including: the creation of tensionswithin the pod between the lignified valve edge cells of the endocarpand the unlignified dehiscence zone (DZ) cells, weakening of the DZ cellwalls by hydrolytic enzyme activity and ultimately due to physicalforces such as strong winds or harvesting machinery.

[0003] Pod development in B. napus can be segmented into three stages.In the first stage, which occurs 0-20 DAA, the newly formed siliques,consisting of two seed-containing carpels separated by a false septumand a replar region, grow to their full length of around 10 cm. Theseeds begin to grow when the pods are virtually full size [Hocking andMason, 1993]. Between 10 and 20 DAA the cells in the replar region beginto differentiate into replar cells, large valve edge cells and form adistinct region, 1-3 cells wide, comprising the DZ [Meakin and Roberts,1990a].

[0004] The second stage occurs between 20 and 50 DAA. From 20 DAA, inconjunction with termination of pod elongation, secondary cell wallmaterial is deposited in the valve edge cells, and the replar cellsbecome increasingly lignified. The DZ cells do not exhibit thickening ofthe cell wall. A progressive shrinkage and loss of organelles isapparent in the DZ cells from 40 DAA onwards and eventually these cellsseparate completely due to hydrolysis of the middle lamella [Meakin andRoberts, 1990a]. In the final stage of pod development, which occurs50-70 DAA, the lignified cells undergo senescence and the necessarytensions are created so that the desiccated pod, containing mature seed,eventually shatters.

[0005] Molecular studies of the penultimate stage of pod developmenthave revealed a spatial and temporal correlation between theup-regulation of a number of mRNAs and pod dehiscence in B. napus. ThesemRNAs encode a polygalacturonase (PG) and a proline-rich protein(SAC51). Further analysis of the expression of the PG following fusionof a pod-specific Arabidopsis thaliana PG promoter to GUS [Jenkins etal., (1997)], has revealed that reporter gene expression is restrictedprecisely to the layer of cells comprising the pod DZ in transgenic B.napus. From 40 DAA, Meakin and Roberts (1990b) reported a progressiveincrease in β-1,4-glucanase (cellulase) activity in the DZ.

[0006] It is understood that the processes of dehiscence and abscissionare not regulated by the same environmental or chemical signals, butthat they involve controlled degradation of cell wall material and cellseparation in a distinct group of cells. Both ethylene andindole-3-acetic acid (IAA) appear to be important regulators of thetiming of the abscission process but the role of these plant hormones indehiscence is less clearly defined. The increase in cellulase activityhas been shown to correlate with a rise in the production of ethylene,mainly from the seed, which peaks at around 40 DAA [Meakin and Roberts,1990b; Johnson-Flanagan and Spencer, 1994].

[0007] Developmental processes, such as pod dehiscence, which involvehighly regulated and controlled expression of an array of differentgenes at a precise time and cellular location, clearly require anintricate signal transduction network.

[0008] Further and improved genetic elements to control plant processesin this area are constantly desired. We describe the isolation, for thefirst time, of a plant cDNA (DZ2) encoding an individual responseregulator protein, the expression of which is closely correlated withdehiscence of fruit in B. napus. DZ2 has a role in the ability tocontrol molecule signaling during the events leading to shatter and thusto control pod shatter in plants. In addition to the identification ofthe nucleic acid termed “DZ2” a homologous, but not identical sequenceand protein were also identified from B. napus. This sequence wasdesignated “DZ2B”. Sequence analysis of DZ2 and DZ2B shows that thereare two DZ2 genes in B. napus, each represented by a slightly differentcDNA (here termed DZ2 and DZ2B). This is consistent with one gene beingencoded by the B. campestris derived genome and the other from thegenome derived from B. oleracea. In this text, the designation “DZ2” isequivalent to the CW1 designation in UK 9806113.8 (as seen from FIG. 1).

[0009] According to a first aspect of the invention there is providednucleic acid optionally encoding a signal transduction protein involvedin the process of dehiscence. Such a sequence or signal transductionprotein has never previously been described in plant dehiscence.

[0010] In this text, the term “involved in the process of dehiscence”means any nucleic acid (preferably) encoding any protein which has aneffect in the dehiscence process, in particular a protein or nucleicacid sequence involved in an MAP Kinase cascade or any other protein ornucleic acid sequence which results in changes in the expression ofgenes involved in dehiscence, such as upregulation of genes encodingpolygalacturonase, cellulase, senescence-related proteins and/ordownregulation of genes encoding for proteins involved in cells wallbiosynthesis. The nucleic acid sequences/proteins of the presentinvention which are “involved in the process of plant dehiscence” arenot the individual structural genes or proteins which cause dehiscence(polygalacturonases etc.). Rather, the nucleic acid sequences/proteinsof the present invention are sequences/proteins which have an effect onthe expression of such structural genes or proteins. One advantage ofthe present invention is that the use of such nucleic acidsequences/proteins enables the possibility to influence the wholeprocess of dehiscence rather than just one element of it. Preferably theprotein or nucleic acid sequence of the present invention which isinvolved in the process of dehiscence effects a structural protein whichis a hydrolytic enzyme such as polygalacturonase or cellulase.

[0011] The nucleic acid of the first aspect of the invention may be anucleic acid which is naturally expressed in a dehiscence zone. Such anucleic acid will most accurately reflect nucleic acid naturallyexpressed in a plant. Preferably the dehiscence zone is a pod (alsotermed “siliques”), anther and/or funiculus dehiscence zone. Preferablythe plant is a member of the Brassica family, maize, wheat, soyabean,Cuphea or sesame.

[0012] A second aspect of the invention provides-nucleic acid encoding aprotein wherein the protein:

[0013] a) comprises the amino acid sequence shown in FIG. 1 or;

[0014] b) has one or more amino acid deletions, insertions orsubstitutions relative to a protein as defined in a) above, but has atleast 40% amino acid sequence identical therewith; or

[0015] c) is a fragment of a protein as defined in a) or b) above, whichis at least 10 (preferably 20 or 30) amino acids long.

[0016] The percentage amino acid identity can be determined using thedefault parameters of the GAP computer program, version 6.0 described byDeveraux et al., 1984 and available from the University of WisconsinGenetics Computer Group (UWGCG). The GAP program utilises the alignmentmethod of Needleman and Wunsch 1970 as revised by Smith and Waterman1981. More preferably the protein has at least 45% identity to the aminoacid sequence of FIG. 1, through 50%, 55% 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% identity using the default parameters.

[0017] The skilled person will appreciate that various changes cansometimes be made to the amino acid sequence of a protein (which has adesired property) to produce variants (often known as “muteins”) whichstill have said property. Such variants of the protein describe in a, band c above are within the scope of the present invention and arediscussed in greater detail below in sections (i) to (iii). They includeallelic and non-allelic variants.

[0018] (i) Substitutions

[0019] An example of a variant of the present invention is a polypeptideas defined in a, b or c above, apart from the substitution of one ormore amino acids with one or more other amino acids.

[0020] The skilled person is aware that various amino acids have similarcharacteristics. One or more such amino acids of a protein can often besubstituted by one or more other such amino acids without eliminating adesired property of that protein.

[0021] For example, the amino acids glycine, alanine, valine, leucineand isoleucine can often be substituted for one another (amino acidshaving aliphatic side chains). Of these possible substitutions it ispreferred that glycine and alanine are used to substitute for oneanother (since they have relatively short side chains) and that valine,leucine and isoleucine are used to substitute for one another (sincethey have larger aliphatic side chains which are hydrophobic). Otheramino acids that can often be substituted for one another includephenylalanine, tyrosine and tryptophan (amino acids having aromatic sidechains); lysine, arginine and histidine (amino acids having basic sidechains); aspartate and glutamate (amino acids having acidic sidechains); asparagine and glutamine (amino acids having amide sidechains); and cysteine and methionine (amino acids having sulphurcontaining side chains).

[0022] Substitutions of this nature are often referred to as“conservative” or “semi-conservative” amino acid substitutions.

[0023] (ii) Deletions

[0024] Amino acid deletions can be advantageous since the overall lengthand the molecular weight of a polypeptide can be reduced whilst stillretaining a desired property. This can enable the amount of proteinrequired for a particular purpose to be reduced. Proteins accordingto(the present invention, which have such deletion(s) are useful. Theymay interfere with the normal functioning-of DZ2; that is, they may actas dominant negative mutations preventing normal DZ2 functioning andthus be of particular value, for example, in reducing pod shatter.

[0025] The amino acid sequence shown in FIG. 1 has various functionalregions. For particular applications of the present invention, one ormore of these regions may not be needed and may therefore be deleted.

[0026] (iii) Insertions

[0027] Amino acid insertions relative to a polypeptide as defined in a,b or c above can also be made. This may be done to alter the nature ofthe protein (e.g. to assist in identification, purification, orexpression, as explained below in relation to fusion proteins).

[0028] Changes in the protein according to the present invention canproduce versions of the protein that are constitutively active. If aprotein of the present invention acts on an inhibitor of the release ofhydrolytic enzymes, then a constitutively active version would preventor reduce pod shatter

[0029] A protein according to any aspect of the invention may haveadditional N-terminal and/or C-terminal amino acid sequences. Suchsequences can be provided for various reasons. Techniques for providingsuch sequences are well known in the art. They include usinggene-cloning techniques to ligate together nucleic acid moleculesencoding polypeptides or parts thereof, followed by expressing apolypeptide encoded by the nucleic acid molecule produced by ligation.

[0030] Additional sequences may be provided in order to alter thecharacteristics of a particular polypeptide. This can be useful inimproving expression or regulation of expression in particularexpression systems. For example, an additional sequence may provide someprotection against proteolytic cleavage; This has been done for thehormone somatostatin by fusing it at its N-terminus to part of the βgalactosidase enzyme [Itakwa et al., 105-63 (1977)].

[0031] Additional sequences can also be useful in altering theproperties of a polypeptide to aid in identification or purification.

[0032] For example, a signal sequence may be present to direct thetransport of the polypeptide to a particular location within a cell orto export the polypeptide from the cell. Hydrophobic sequences may beprovided to anchor a polypeptide in a membrane. Thus the presentinvention includes within its scope both soluble and membrane-boundpolypeptides.

[0033] Preferably, the nucleic acid according to the second aspect ofthe invention encodes a signal transduction protein or a functionalportion thereof involved in the process of dehiscence. All preferredfeatures of the first aspect of the invention as described above alsoapply to the second.

[0034] The term protein used in this text means, in general terms, aplurality of amino acid residues joined together by peptide bonds. It isused interchangeably and means the same as polypeptide or peptide.

[0035] The nucleic acid according to the first or second aspect of theinvention preferably comprises the sequence set out in FIG. 1 or asequence which is 40% or more identical, preferably through 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% to the sequence in FIG. 1 atthe nucleic acid residue level, using the default parameters of the GAPcomputer program, version 6.0 described by Deveraux et al., 1984 andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilises the alignment method of Needleman andWunsch 1970 as revised by Smith and Waterman 1981. Further, the nucleicacid may comprise a fragment of a sequence according to the first orsecond aspect which is at least 30 bases long also 40, 50, 60, 70, 80,or 90 bases in length. While this nucleic acid is the preferred nucleicacid of the invention, it is well known to those persons skilled in theart that because of the nucleic acid “degenerate code” which encodesnucleic acids, a considerable number of variations in nucleic acidsequence can be used to encode for proteins according to the first orsecond aspects of the invention.

[0036] The nucleic acid of the first or second aspects of the inventionmay be isolated or recombinant and may be in substantially pure form.The nucleic acid may be antisense to nucleic acid according to the firstor second aspects of the invention. As understood by the person skilledin the art introducing the coding region of a gene in the reverseorientation to that found in nature (antisense) can result in thedownregulation of the gene and hence the production of less or none ofthe gene product. The transcribed antisense DNA is capable of binding toand destroying the function of the sense RNA of the sequence normallyfound in the cell, thereby, disrupting function. Antisense nucleic acidmay be constitutively expressed, but is preferably limited to expressionin those zones (dehiscence) in which the naturally occurring nucleicacid is expressed.

[0037] The nucleic acid according to the first or second aspects of theinvention preferably include a promoter or other regulatory sequencewhich controls expression of the nucleic acid. Promoters and otherregulatory sequences which control expression of a nucleic acid indehiscence zones are known in the art, for example described inW096/30529 and W094/23043. Further promoters or other regulatorysequences can be identified and can also include the promoter or otherregulatory sequence which controls expression of a nucleic acid as setout in FIG. 1. The person skilled in the art will know that it may notbe necessary to utilize the whole promoter or other regulatory sequence.Only the minimum essential regulatory elements may be required and infact such elements can be used to construct chimeric sequences orpromoters. The essential requirement is, of course, to retain the tissueand/or temporal specificity.

[0038] The nucleic acid according to the first or second aspects of theinvention may be in the form of a vector. The vector may be a plasmid,cosmid or phage. Vectors frequently include one or more expressedmarkers which enable selection of cells transfected (or transformed: theterms are used interchangeably in this text) with them and preferably,to enable a selection of cells containing vectors incorporatingheterologous DNA. A suitable start and stop signal will generally bepresent and if the vector is intended for expression, sufficientregulatory sequences to drive expression will be present. Nucleic acidaccording to the first and second aspects of the invention is preferablyfor expression in plant cells and thus microbial host expression isperhaps less important although not ruled out. Microbial host expressionand vectors not including regulatory sequences are useful as cloningvectors.

[0039] A third aspect of the invention relates to a cell comprisingnucleic acid according to the first or second aspects of the invention.The cell may be termed as “a host” which is useful for manipulation ofthe nucleic acid, including cloning. Alternatively, the cell may be acell in which to obtain expression of the nucleic acid, most preferablya plant cell. The nucleic acid can be incorporated by standardtechniques known in the art in to cells. Preferably nucleic acid istransformed in to plant cells using a disarmed Ti plasmid vector andcarried by an Agrobacterium by procedures known in the art, for exampleas described in EP-A-0116718 and EP-A-0270822. Foreign nucleic acid canalternatively be introduced directly into plant cells using anelectrical discharged apparatus or by any other method that provides forthe stable incorporation of the nucleic acid into the cell. Preferablythe stable incorporation of the nucleic acid is within the nucleic DNAof any cell preferably a plant cell. Nucleic acid according to the firstand second aspects of the invention preferably contains a second“marker” gene that enables identification of the nucleic acid. This ismost commonly used to distinguish the transformed plant cell containingthe foreign nucleic acid from other plants cells that do not contain theforeign nucleic acid. Examples of such marker genes include antibioticresistance, herbicide resistance and Glucuronidase (GUS) expression.Expression of the marker gene is preferably controlled by a secondpromoter which allows expression of the marker gene in cells other thanthose than dehiscence zones (if this is the tissue specific expressionof the nucleic acid according to the first or second aspects of theinvention). Preferably the cell is from any of the Brassica family (mostpreferably B. napus), maize, wheat, soyabean, Cuphea and sesame.

[0040] A third aspect of the invention includes a process for obtaininga cell comprising nucleic acid according to the first or second aspectsof the invention. The process involves introducing said nucleic acidinto a suitable cell and optionally growing on or culturing said cell.

[0041] A fourth aspect of the invention provides a plant or a partthereof comprising a cell according to the third aspect of theinvention. A whole plant can be regenerated from the single transformedplant cell by procedures well known in the art. The invention alsoprovides for propagating material or a seed comprising a cell accordingto the third aspect of the invention. The invention also relates to anyplant or part thereof including propagating material or a seed derivedfrom any aspect of the invention. The fourth aspect of the inventionalso includes a process for obtaining a plant or plant part (includingpropagating material or seed, the process comprising obtaining a cellaccording to the third aspect of the invention or, indeed, plantmaterial according to the fourth aspect of the invention and growth (tothe required plant, plant part, propagating material etc). Techniquesfor such a process are commonplace in the art.

[0042] A fifth aspect of the invention provides a signal transductionprotein involved in the process of the plant dehiscence. The signaltransduction protein according to the fifth aspect may have one or moreof the preferred features according to the first or second aspects ofthe invention. Preferably it may be isolated, recombinant or insubstantially pure form. It may comprise the various changes accordingto the first or second aspects. Preferably the protein is expressed fromnucleic acid according to the first or second aspects. Alternatively,the protein can be provided using suitable techniques known in the art.

[0043] A sixth aspect of the invention provides a protein which;

[0044] a) comprises the amino acid sequence shown in FIG. 1 or;

[0045] b) has one or more amino acid deletions, insertions, orsubstitutions relative to a protein as defined in a) above and has atleast 40% amino acid sequence identity therewith;

[0046] or a fragment of a protein as defined in a) or b) above which isat least 10 amino acids long. The percentage amino acid identity can bedetermined using the default parameters of the GAP computer program,version 6.0 described by Deveraux et al., 1984 and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The GAP programutilises the alignment method of Needleman and Wunsch 1970 as revised bySmith and Waterman 1981. More preferably the protein has at least 45%identity to the amino acid sequence of FIG. 1, through 50%, 55% 60%,65%, 70%, 75% 80%, 85%, 90%, 95% identity using the default parameters.

[0047] The protein is preferably a signal transduction protein involvedin the process of plant dehiscence and again, the preferred features ofaspects one, two and five also applied to the sixth aspect.

[0048] The seventh aspect of the invention provides a process forregulating/controlling dehiscence in plant or in a part thereof, theprocess comprising obtaining a plant or a part thereof according to thefourth aspect of the invention. The process of dehiscence can beregulated and/or controlled by increasing or decreasing the expressionof nucleic acid sequences according to the first or second aspect of theinvention. Increased or decreased expression can easily be influenced bythe person skilled in the art using technology well known. This includesincreasing the numbers of copies of nucleic acid according to theinvention in a plant or a plant thereof or increasing expression levelsof copies of the nucleic acid present in particular parts or zones ofthe plant. Preferably the zones are dehiscence zones. The processaccording to the seventh aspect of the invention includes obtaining aplant cell according to the third aspect of the invention or part of aplant according to the fourth aspect in the invention and deriving aplant therefrom. Alternatively, the process may comprise obtainingpropagating material or a seed according to the fourth aspect of theinvention and deriving a plant therefrom.

[0049] Preferably, the process of the seventh aspect of the invention isin the pod or the anther of a plant. All preferred features of aspectsone to six also apply to the seventh.

[0050] An eighth aspect of the invention provides for the use of nucleicacid according to the first to seventh aspects of the invention in theregulation/control of plant dehiscence. All preferred features ofaspects one to seven also applies to the eighth.

[0051] The ninth aspect of the invention provides for the use of nucleicacid according to the first or second aspect of the invention as aprobe. Such a probe can be used in techniques well known in the art toidentify the presence of identical or homologous nucleic acid sequencesfrom any source, preferably a plant source. The ninth aspect of theinvention also provides nucleic acid identified by use of the nucleicacid from aspects one or two as a probe.

[0052] A tenth aspect of the invention provides for the use of nucleicacid according to aspects one or two of the invention in the productionof a cell, tissue, plant or part thereof, or propagating material.Again, all preferred features of aspects one and two also apply to thetenth.

[0053] An eleventh aspect of the invention provides for nucleic acidcomprising one or more of the underlined sequences as set out in FIG. 1or the primer sequences in FIG. 5, FIG. 9 or FIG. 11. Such nucleic acidsequences are preferably used as primers in an PCR (Polymerase ChainReaction) process in order to amplify nucleic acid sequences.

[0054] A twelfth aspect of the invention provides the use of nucleicacid according to the first or second aspects of the invention toidentify another other protein or proteins which interact with itsexpression product. Such use can be carried out by the yeast two hybridscreening method (or others known in the art). The yeast two hybridscreening method is described for this aspect of the invention, ingeneral, with reference to the sequence described as DZ2. A potentialway to implement the yeast 2-hybrid screen is outlined, as follows:

[0055] DZ2 is linked to the Gal4 DNA binding domain and expressed inyeast which contains a pGAL4-lacZ gene. For activity of lacZ a secondprotein is required that contains the DNA transcriptional activationdomain of GAL4 and that interacts with the DZ2 protein. This is providedby making a cDNA expression library from plant DZ zones which results infusions of plant proteins to the GAL4 activation domain. This library istransformed into the yeast strain that contains pGAL4-LacZ and expressesthe DZ2-Gal4 DNA bining domain protein fusion. Colonies that have lacZactivity are transformed with a gene for a protein that interacts withDZ2.

[0056] Using such a system, upstream and downstream components of anysignal transduction pathway can be identified, thus resulting in furtherability to control/regulate dehiscence and/or male sterility.

[0057] A thirteenth aspect of the invention provides for a protein, asdefined according to the limitations of the second aspect of theinvention (without reference to FIG. 1) and nucleic acid encoding theprotein, wherein the protein is capable of being identified according tothe use (or method) according to the twelfth aspect of the invention.

[0058] A fourteenth aspect of the invention provides for the use of aprotein according to the fifth or sixth aspect of the invention as aprobe. In this context the probe is a means to identifying interactingentities (such as other proteins), including upstream and downstreaminteracting signal components. A protein according to the fifth or sixthaspect of the invention can be used as a probe to directly look forinteractions with other proteins, i.e. purified protein can be used tolook for complex formation with other plant protein, particularlyisolated from the DZ zone. For example, a modified recombinant DZ2protein can be made with a sequence tag, such as a His-tag, that enablesthe DZ2+interacting protein to be directly purified on a His affinitycolumn. Alternatively, an antibody can be raised to DZ2 protein. Thisantibody is then used to identify DZ2 protein complexes and to purifythe complexes. The DZ2 interacting proteins can be purified andmicrosequenced to enable cloning of the genes for these interactingproteins.

[0059] The present invention provides a particularly useful method bywhich plant dehiscence can be regulated/controlled.

[0060] In addition to the use of the present invention in the productionof shatter resistance or shatter-delayed plants such as oil seed rape,the invention may be used to control/regulate pollen release (by thecontrol/regulation of anther dehiscence) which can produce male sterileplants. The temporal and spatial expression of nucleic acid encoding aprotein according to the first and second aspects of the invention mayrequire adjustment in obtaining the correct levels of dehiscence delayor prevention in different zones. For example, if pod dehiscence isrequired but anther dehiscence is not, it is necessary to ensure thatexpression of nucleic acid according to a first or second aspect of theinvention has the correct temporal and spatial expression in order toobtain pod dehiscence or delay but not, to any substantial extent,anther dehiscence. This can be obtained by processes known in the artand may require use of particular promoter sequences to obtain thedesired result. Usually in plant transformation, some difference in thelevel of expression of nucleic acid is observed in different plants. Insome cases, the ratio of expression levels in different tissues can varybetween different plant transformants thus providing essentiallytissue-specific expression in one or other of the target tissues in someof the plant transformants. In the present invention, the naturalexpression of nucleic acid according to the first or second aspects maybe predominantly higher in pod dehiscence zones and lower in the antherand funiculus dehiscence zones. However, as described above, it ispossible to obtain plants in which the protein expression is regulatedin a particular dehiscence zone. Accordingly, a particularly usefulaspect of the invention is the provision of plants which have one orboth of the following features; are male sterile, are shatter resistant.

[0061] As described earlier, the process of dehiscence at the dehiscencezone involves the secretion of a number of enzymes, including hydrolyticenzymes. While previous attempts have been made to down or up regulatespecific genes encoding particular proteins involved in the process ofdehiscence, regulation by means of a signal transduction protein whicheffects expression of a number of genes is likely to be more effectivethan regulation of a single gene. In addition to this, the nucleic acidof the present invention has been identified as being expressed earlierthan several other known genes involved in the process of plantdehiscence. This suggests that it is important earlier on in the processof plant dehiscence and can be used to control/regulate plant dehiscenceat an earlier stage.

[0062] The nucleic acid encoding a signal transduction protein involvedin the process of dehiscence or the signal transduction protein itselfmay be a component of a signal pathway that may either positively ornegatively regulate pod shatter.

[0063] A more detailed explanation of such regulations/control,described with reference to a pod shatter (dehiscence) model isdescribed below. As a skilled person will acknowledge, the modeldescribed below also relates to other general processes of dehiscencesuch as in the anther.

[0064] In the process of dehiscence, a particular signal transductionprotein may be required to transmit a signal from the almost mature seedwhich initiates the expression or release of enzymes required for podshatter. In this model, developmental signals switch on expressionand/or activation of a particular signal transduction protein in the poddehiscence zone. This leads to expression of genes required for therelease of pod dehiscence zone enzymes (such as hydrolytic enzymes). Inthis case, prevention of activity of the signal transduction protein,for example by downregulation of expression of this protein, wouldresult in reduced dehiscence.

[0065] Alternatively, the developing seed may transmit a signal whichrepresses the expression and/or activity of a particular signaltransduction protein until late in cell development. In this model,developmental signals switch on a particular signal transduction proteinwhich, in due course, represses the expression of genes required forrelease of dehiscence zone specific enzymes (such as hydrolyticenzymes). In this case, expression of a modified signal transductionprotein that is constitutively active would result in reduceddehiscence.

[0066] A signal transduction protein which is either positively ornegatively involved in the process of dehiscence can be used accordingto the present invention.

[0067] In addition to DZ2 several other DZ-expressed genes have beenpreviously isolated and individually downregulated to result in B. napusplants that have increased resistance to pod shatter; namely Sac66 (WO96/30529—FIG. 15), DZ15 (FIG. 16) and OSR 7(9) (FIG. 17). It isanticipated that downregulation of more than one gene involved in podshatter will further increase resistance to pod shatter. This could beachieved by combining different transgenes by transformation withseveral transgenes each designed to downregulate a differentDZ-expressed gene or by crossing together B. napus lines thatindividually are transformed with such transgenes. Such methods arecomplex either involving transformation with a construct containingmultiple chimeric genes or require the maintenance of several transgenicloci in the breeding program. A preferred method is to transform with achimeric gene consisting of a single promoter driving expression of anantisense or partial sense transcript which is comprised of elements ofall the DZ-expressed genes to downregulated. Similarly a single promotercould be used to drive the expression of multiple ribozymes eachtargeted against a different DZ-expressed gene. The use of a singlepromoter to drive expression of a combination of antisense, partialsense and ribozymes is also possible. Ideally the promoter will be podDZ-specific, however a useful promoter may be pod-specific or evenconstitutively active. A suitable DZ-specific promoter would be that ofDZ2, DZ2B, DZ2AT3 or ESJ2A (WO 99/13089).

[0068] Accordingly, the present invention provides a nucleic acidsequence according to the first or second aspects (and also all aspectswhich include the first or second aspects) in combination with one ormore further nucleic acid sequences which are dehiscence-zone expressed.Examples of such sequences include Sac66, DZ15 and OSR(7), FIGS. 15-17respectively. Such sequence may be in sense or in antisense orientation.Such a sequence may be included as full length genomic, full-length cDNAor partial sequences; the sequences may be as shown in the figures ormay have the same sequence identity (both for aminoacid sequence andnucleic acid sequence) as described above for the protein according tothe second aspect of the invention or the nucleic acid according to thefirst or second aspects of the invention. As will be recognised by thoseskilled in the art a partial sequence may be useful in either the senseor antisense orientation.

[0069] The invention is described by reference to the enclosed drawings;

[0070]FIG. 1 DZ2 full length sequence-showing original PCR product andprimer sites

[0071]FIG. 2 Amino acid alignment with bacterial response regulatorproteins & EYR1

[0072]FIG. 3 Northern analysis of expression of DZ2 in pods and othertissues. The lower panel shows the ethidium bromide-stained RNA gelprior to blotting and probing with DZ2

[0073]FIG. 4 Comparison of bacterial two-component systems with DZ2

[0074]FIG. 5 Sequence of the promoter region of B.napus DZ2B.

[0075]FIG. 6 Nucleic and putative peptide sequence alignments of DZ2with DZ2B.

[0076]FIG. 7 Northern analysis of expression of DZ2B in pods and othertissues. The probe was labeled DZ2B cDNA.

[0077]FIG. 8 Schematic diagram of pDZ2B-GUS-SCV

[0078]FIG. 9 DZ2AT3 cDNA sequence showing the putative DZ2AT3 peptide.

[0079]FIG. 10 Amino acid alignment of DZ2AT3 with DZ2 and DZ2B.

[0080]FIG. 11 Sequence of the promoter region of A.thaliana DZ2AT3.

[0081]FIG. 12 Schematic diagram of pDZ2AT3GUS-SCV.

[0082]FIG. 13 Schematic diagram of pPGL-DZ2as-SCV and pDZ2B-DZ2as-SCV.

[0083]FIG. 14 Schematic diagram of pWP357-SCV.

[0084] Table 1 Pod shatter resistance of WP357-SCV transformants.

[0085]FIG. 15 Nucleic acid sequence and putative amino acid sequence ofSac66.

[0086]FIG. 16 Nucleic acid sequence and putative amino acid sequence ofDZ15.

[0087]FIG. 17 Nucleic acid sequence and putative amino acid sequence ofOSR7 (9)

[0088] The present invention is now described with reference to thefollowing, non-limiting examples.

EXAMPLE 1 Isolation and Characterisation of Expression of DZ2 PlantMaterial

[0089] Seeds of B. napus cv Rafal were grown as described by Meakin andRoberts, (1990a) with the following modifications. Single seedlings wereported into 10 cm pots, and after vernalization, were re-potted into 21cm pots. At anthesis tags were applied daily to record flower opening.This procedure facilitated accurate age determination of each pod. Podswere harvested at various days after anthesis (DAA). The dehiscence zonewas excised from the non-zone material and seed using a scalpel blade(Meakin and Roberts (1990b)) and immediately frozen in liquid No andstored at −70° C.

RNA Isolation

[0090] All chemicals were molecular biology grade and bought from eitherSigma Chemical Ltd (Dorset, UK), or Fisons (Loughborough, UK). Total RNAwas extracted using the polysomal extraction method of Christoffersenand Laties, (1982), with the following alterations. The plant materialwas ground to a powder in liquid N, and then in 10 volumes of extractionbuffer (200 mM Tris-acetate [pH 8.2], 200 mM magnesium acetate, 20 mMpotassium acetate, 20 mM EDTA, 5% w/v sucrose, after sterilisation2-mercaptoethanol was added to 15 mM and cycloheximide added to a finalconcentration of 0.1 mg ml⁻¹). The supernatant was then layered over 8ml 1M sucrose made with extraction buffer and centrifuged in a KONTRON™(Switzerland) TFT 70.38 rotor at 45,000 rpm (150,000 g) for 2 hr at 20°C. in a Kontron CENTRIKON™ T-1065 ultra-centrifuge. Pellets were thenresuspended in 500 μl 0.1M sodium acetate, 0.1% SDS, pH 6.0 andphenol/chloroform (1:1 v/v) extracted and the total RNA precipitated.Poly(A)⁺ RNA was isolated from total RNA extracted, from both the zoneand non-zone tissue of 40, 45 and 50 DAA pods, using a Poly(A) QUIK™mRNA purification kit (Stratagene, Cambridge, UK) following themanufacturers instructions, and then bulked together. Total RNA was alsoextracted from leaves, stems, seeds and pods using a method described byDean et al, (1985) for use in Northern analyses.

[0091] Differential Display

[0092] This was performed essentially as described by Liang and Pardee(1992) using RNA extracted from 40 DAA pod dehiscence zones andnon-zones. First strand cDNA copies of the RNAs (40 DAA DZ/NZ) were madeusing 50U M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase(50U/μL) (Stratagene) in a 20 μL reaction containing 1×M-MLV buffer, 2.5mM dNTPs (Pharmacia), 1 μg RNA, 30U RNAse inhibitor (Promega) and 10 μMoligo dT anchor primer 7 (5′-TTTTTTTTTTTTGG-3′). The reaction conditionswere as follows: 65° C. for 5 minutes, 37° C. for 90 minutes and 95° C.for 5 minutes. Following first strand cDNA synthesis, 60 μL dH20 wereadded and the samples were either used directly for PCR or stored at−20° C.

[0093] For PCR, 2 μL cDNA were used as template in a 20 μL reactioncontaining 1×PCR buffer, 1 mM MgCl₂, 2 μM dNTPs, 10 μM oligo dT anchorprimer 7 (5′-TTTTTTTTTTTTGG-3′), 2.5 μM arbitrary primer A (5′-AGC CAGCGA A -3′), 0.5 μL 35S-dATP (>1000 Ci/mmol) (Amersham) and 1U Taq DNApolymerase (5U/μL) (Gibco BRL). The thermocycling conditions were asfollows: 40 cycles of 94° C. for 30 seconds, 40° C. for 2 minutes, 72°C. for 30 seconds followed by 72° C. for 5 minutes. The PCR productswere fractionated on a 5% polyacrylamide/7M urea gel after addition of 5μL loading buffer (95% (v/v) formamide, 20 mM EDTA, 0.05% (w/v) xylenecyanol, 0.05% (w/v) bromophenol blue) to each sample. Followingelectrophoresis the gel was dried at 80° C. under vacuum for 1 hour thenexposed to X-ray film (BioMax-MR, Kodak) in a light tight cassette for48 hours. The dried gel and autoradiogram were aligned so that bandsthat appeared in the DZ and not in NZ could be cut out and the DNAeluted according to Liang et al. (1995). The eluted PCR products (4 μL)were reamplified in a 40 μL reaction containing 1×PCR buffer, 1 nMMgCl₂, 20 μM dNTPs, 10 μM oligo dT anchor primer 7(5′-TTTTTTTTTTTTGG-3′), 2.5 μM arbitrary primer A (5′-AGC CAG CGA A-3′)and 2U Taq DNA polymerase (5U/μL) (Gibco BRL) using the followingthermocycling conditions. 40 cycles of 94° C. for 30 seconds, 40° C. for2 minutes, 72° C. for 30 seconds followed by 72° C. for 5 minutes. Theresulting PCR product was cloned into the TA cloning vector (Invitrogen)and sequenced (FIG. 1). In order to prepare an antisense strand-specificriboprobe, the PCR product was subcloned into pBluescript (Stratagene).

[0094] Expression Analysis and Characterisation of DZ2

[0095] Northern analysis using an antisense strand-specific riboprobe tothe DZ2 PCR product, showed that DZ2 hybridised to a transcript of 0.6kb which is expressed in the DZ of 20-50 DAA pods with a peak inexpression at 40DAA. Minimal expression was observed in the pod NZ [FIG.2]. A random-primed labelled DNA probe (Stratagene) of the 330 bp DZ2PCR product (amplified using primers DZ2FL and DZ2RL—see FIG. 1) wasused to screen a B. napus DZ cDNA library from which, following threerounds of screening to obtain pure plaques, a full length DZ2 cDNA (606bp) was obtained (FIG. 1). An antisense strand-specific riboprobe of thefull length DZ2 cDNA was hybridised to total RNA extracted from podDZ/NZ (as in FIG. 2), leaf abscission zones (AZ) and non-zones (NZ)(following exposure to 10 μL/L ethylene for 72 hours), seed, root,flower and leaf. FIG. 3 shows that DZ2 hybridises to a 0.6 kb messagewhich is present in the pod DZ at 20-50 DAA with maximum expression at40DAA. Again there is minimal expression in pod NZ and no apparentexpression of DZ2 in AZ, NZ, leaf, root, seed or flower RNA. By thesensitive technique of RT-PCR analysis DZ2 expression can also bedetected in anthers and the funiculus, both tissues that containdehiscent zones

[0096] The 606 bp cDNA (DZ2) encodes a putative protein of 136 aminoacids. Comparison of the DZ2 translated sequence to the OWL proteindatabase [Bleasby and Attwood (1994)] showed low but consistent homologyto a group of bacterial proteins comprising two-component regulatorysystems. In particular, DZ2 possesses the conserved amino acid residuesrequired for phosphorylation of the receiver domain of the responseregulator component (see FIG. 4). DZ2 plays a role in a signaltransduction cascade resulting at least in one respect in pod shatter.It is therefore a good candidate for down-regulation of pod shatterprocesses using antisense technology. DZ2 is a novel plant protein inthat independent proteins with homology to bacterial receivers are yetto be reported in plants.

[0097] The full length cDNA was excised from the pBluescript cloningvector by digestion with EcoRI and XhoI restriction enzymes (Gibco BRL).Following purification from a 1% agarose gel the 606 bp cDNA was randomprimed labelled (Stratagene) and used to screen a B. napus genomiclibrary in the BlueStar vector. Following three rounds of screening toobtain pure plaques, a single genomic clone was isolated which carries a15 kb genomic DNA insert. The promoter of the DZ2 gene is isolated fromthis genomic clone using standard techniques (see Example 2).

EXAMPLE 2 Isolation and Characterisation of the B.napus DZ2B Promoter.

[0098] To obtain the B.napus DZ2 promoter a B.napus genomic library wasscreened with a labelled DZ2 probe. The full length cDNA was excisedfrom the pBluescript cloning vector (Stratagene) by digestion with EcoRIand XhoI restriction enzymes (Gibco BRL). Following purification from a1% agarose gel the 606 bp cDNA was random primed labelled (Stratagene)and used to screen a B. napus genomic library in the BlueStar vector(Novagen). Following three rounds of screening to obtain pure plaques, asingle genomic clone was isolated which carries a 15 kb genomic DNAinsert. The region hybridising to DZ2 was sequenced and found to encodea protein homologous to, but not identical to DZ2. This DZ2-like genewas designated DZ2B (FIG. 5). The primers DZ2BFL (FIG. 5) and T7 wereused to PCR out a DZ2B cDNA from the B.napus DZ cDNA library. 5′AACCAAGTCAGTAGAAGTG 3′ DZ2BFL 5′ AATACGACTCACTATAGG 3′ T7

[0099] The DZ2 and DZ2B cDNAs are 80% identical (according to thedefault parameters of the GAP computer program, version 6, Deveraux etal., 1984, and available from the University of Winsconsin GeneticsComputer Group (UWGCG)) at the nucleotide level in the region of overlapof the coding sequences (FIG. 6a) and the putative proteins encoded byDZ2 and DZ2B are 80% identical (according to the default parameters ofthe GAP computer program, version 6, Deveraux et al., 1984, andavailable from the University of Winsconsin Genetics Computer Group(UWGCG)) (FIG. 6b). Sequence analysis. of more DZ2 and DZ2-like cDNAsand Southern analysis shows that there are two DZ2 genes in B.napus, DZ2and DZ2B, each represented by 2 slightly different cDNAs. This isconsistent with one gene being encoded by the B.campestrisderived-genome and the other from the genome derived from B.oleracea.

[0100] RT-PCR with primers specific to DZ2B showed that DZ2B is onlyexpressed in pods. This was confirmed by northern analysis which showedpreferential expression in the DZ (FIG. 7). Thus DZ2B has a similarpattern of expression as DZ2 and is thus a suitable source of aDZ-expressed promoter.

[0101] Primers DZ2BGenF and DZ2BGenR were used to PCR a 1253 bp DZ2Bpromoter fragment (FIG. 5). 5′ GGCTCTAGACGAACTGCGGAGCAAGG 3′ DZ2BGENF 5′CTGCCATGGTCGGTTTTTTTTCTTCGAAC 3′ DZ2BGENR

[0102] These primers introduced an XbaI site at the 5′ end of the PCRfragment and an NcoI site around the initiating Met of DZ2B. Thus thePCR fragment was cloned as an XbaI, NcoI fragment between the XbaI andNcoI sites of pWP272 (WO 99/10389) forming pDZ2B-GUS. The chimericpDZ2B-GUS-CaMV polyA gene was then transferred as an XbaI, XhoI fragmentbetween the XbaI and SalI sites of pSCV nos-nptII-(WO 95/20668) formingpDZ2B-GUS-SCV (FIG. 8). The pDZ2B-GUS-SCV binary vector was.transferredto the agrobacterial strain pGV2260 and transformed B.napus plantsproduced by agrobacterial transformation essentially as described inMoloney M et al., (1989). Gus expression is observed in the pod DZ.

EXAMPLE 3 Isolation and Characterisation of a DZ2 Arabidopsis thalianaHomologue

[0103] To demonstrate that a DZ2 orthologous gene can be isolated fromanother plant species the functional equivalent of B.napus DZ2/DZ2B wasisolated from Arabidopsis thaliana. The B.napus DZ2 cDNA was used as aprobe to screen an Arabidopsis cDNA library (J. Giraudat, ISV-CNRS,France). FIG. 9 shows the sequence of a cDNA (DZ2AT3) that hybridised tothe DZ2 probe. DZ2AT3, has 85% nucleic acid identity to DZ2 and 85% toDZ2B (according to the default parameters of the GAP computer program,version 6, Deveraux et al., 1984, and available from the University ofWinsconsin Genetics Computer Group (UWGCG)) in the coding regions whichare common to all three sequences. The putative peptide encoded byDZ2AT3 has 80% identity to DZ2 and 80% to DZ2B (according to the defaultparameters of the GAP computer program, version 6, Deveraux et al.,1984, and available from the University of Winsconsin Genetics ComputerGroup (UWGCG)) in the regions which are common to all three sequences(FIG. 10). RT-PCR analysis of RNA isolated from leaves, roots, flowersand siliques showed that DZ2AT3 was specifically expressed in siliques.Southern hybridisation analysis showed that the DZ2AT3, DZ2 and DZ2Bprobes each identify a single identical band in A.thaliana. Thisindicates that A.thaliana contains one DZ2 gene in contrast to B.napuswhich contains two.

[0104] The Genome walker kit (Clonetech) was used to isolate the DZ2AT3promoter from A.thaliana genomic DNA. Nested PCR was performed usingprimer GW1 first, then AT3GW2 each in conjunction with the Genome Walkerkit primer (FIG. 9). FIG. 11 shows the sequence of the promoter regionof DZ2AT3 thus obtained. The primers ATDZ2F and ATDZ2R were used to PCRa 1195 bp promoter fragment from the DZ2AT3 genomic sequence (FIG. 11).5′ CACTAGTAGGGCACGCGTGGTCG 3′ ATDZ2F 5′ TCCATGGTCGATTTCTTTTCTCTCAAG 3′ATDZ2R

[0105] These primers introduced an SpeI site at the 5′ end of the PCRfragment and an NcoI site around the initiating Met of DZ2AT3. Thus thePCR fragment was cloned as an SpeI, NcoI fragment between the XbaI andNcoI sites of pWP272 (WO 99/13089) forming pDZ2AT3-GUS. The chimericpDZ2AT3-GUS-CaMV polyA gene was then transferred as a SalI, XhoIfragment into the Sall site of pSCV nos-nptII (WO 95/20668) formingpDZ2AT3-GUS-SCV (FIG. 12). The pDZ2AT3-GUS-SCV binary vector wastransferred to the agrobacterial strain pGV2260 and transformed B.napusplants produced by agrobacterial transformation essentially as describedin Moloney M et al., (1989). Gus expression is observed in the pod DZ.

EXAMPLE 4 Production of Shatter-resistant B.napus Plants by AntisenseDownregulation of DZ2

[0106] Downregulation of the DZ2 gene or reduction in DZ2 protein levelsin the pod DZ will result in plants that are resistant (or moreresistant than without this modification) to pod shatter. Standardtechniques, commonplace in the art, such as the expression of antisenseDZ2 mRNA, full sense mRNA, partial sense mRNA or a ribozymedirected-against DZ2 mRNA are effective. Expression of these RNAsrequires a promoter that is active in the pod DZ at the time at whichDZ2 is expressed. Ideally the promoter will be pod DZ-specific, howevera useful promoter may be pod-specific or even constitutively active. Asuitable promoter would be that of DZ2. Although DZ2 is expressed in theanther DZ, pod DZ and funiculus DZ, DZ2 promoter -GUS fusion studiesshow that in different transformants the relative level of expression inthese three sites is variable but is stability hereditable. Thus sometransformants are obtained in which expression is largely or exclusivelyconfined to the pod DZ. This suggests that the pDZ2 promoter iscomprised of distinct elements each specifying expression in aparticular DZ. Alternatively the site of transgene integration mayinfluence relative expression levels in the DZ tissues. The DZ2 promoteris therefore linked to the DZ2 cDNA such that the DZ2 is in theantisense orientation forming pDZ2-antiDZ2. This chimeric gene istransferred to the binary vector pNos-NptII-SCV (WO 96/30529). Thisbinary vector is transferred to the agrobacterial strain pGV2260 andtransformed B.napus plants produced by agrobacterial transformationessentially as described in Moloney M et al., (1989) Plant Cell Reports8, 238-242. A proportion of transformed B.napus plants exhibit reducedlevels of DZ2 message and are resistant to pod shatter.

EXAMPLE 5 Production of Shatter-resistant B.napus Plants by AntisenseDownregulation of DZ2

[0107] Downregulation of the DZ2 gene or reduction in DZ2 protein levelsin the pod DZ will result in plants that are resistant to pod shatter.Techniques such as the expression of antisense DZ2 mRNA, full sensemRNA, partial sense mRNA or a ribozyme directed against DZ2 mRNA will beeffective. Expression of these RNAs requires a promoter that is activein the pod DZ at the time at which DZ2 is expressed. Ideally thepromoter will be pod DZ-specific, however a useful promoter may bepod-specific or even constitutively active. Although DZ2/DZ2B isexpressed in the anther DZ, pod DZ and funiculus DZ, DZ2B promoter GUSfusion studies show that in different transformants the relative levelof expression in these three sites is variable but is stablyhereditable. Thus some transformants are obtained in which expression islargely or exclusively confined to the pod DZ. This suggests that thepDZ2 promoter is comprised of distinct elements each specifyingexpression in a particular DZ. Alternatively the site of transgeneintegration may influence relative expression levels in the DZ tissues.Thus a suitable DZ-specific promoter would be that of DZ2, DZ2B, DZ2AT3or ESJ2A (WO 99/13089).

[0108] The primers DZ2FLA and DZ2RLA were used to PCR a 349 bp fragmentfrom the DZ2 cDNA: 5′ GGCGAATTCCGGTGAGGAGGCAGTAATC 3′ DZ2FLA 5′GGCCCATGGCATACATACACACTTAGAC 3′ DZ2RLA

[0109] The primers introduce an EcoRI and NcoI site at the ends of theDZ2 PCR fragment. To link the DZ2 PCR fragment in an antisenseorientation to the promoter of ESJ2A (PPGL) the DZ2 PCR fragment wascloned as a NcoI, EcoRI fragment between the NcoI and EcoRI sites ofpWP272 (WO 99/13089) forming pPGL-DZ2as. The pPGL-antisense DZ2 chimericgene was transferred as a XbaI, XhoI fragment from pDZ2as into the XbaIand Sall sites of the binary vector pSCV nos-nptII (WO 95/20668) formingpPGL-DZ2as-SCV (FIG. 13a).

[0110] The pPGL-DZ2as-SCV binary vector was transferred to theagrobacterial strain pGV2260 and transformed B.napus plants produced byagrobacterial transformation essentially as described in Moloney M etal., (1989). A proportion of transformed B.napus plants exhibit reducedlevels of DZ2 and DZ2B message and were resistant to pod shatter

[0111] Similarly, to link the DZ2 PCR fragment in an antisenseorientation to the promoter of DZ2B, the DZ2 PCR fragment is cloned as aNcoI, EcoRI fragment between the NcoI and EcoRI sites of pDZ2B-GUSforming pDZ2B-DZ2as. The pDZ2B-DZ2as chimeric gene is transferred as aXbaI, XhoI fragment from pDZ2B-DZ2as into the XbaI and SalI sites of thebinary vector pSCV nos-nptII (WO 95/20668) forming pDZ2B-DZ2as-SCV (FIG.13b).

[0112] The pDZ2B-DZ2as-SCV binary vector is transferred to theagrobacterial strain pGV2260 and transformed B.napus plants. Again aproportion of transformed B.napus plants exhibit reduced levels of DZ2and DZ2B message and are resistant to pod shatter.

[0113] Similarly a proportion of B.napus plants transformed with apDZ23A-DZ2as-SCV construct exhibit reduced levels of DZ2 and DZ2Bmessage and are resistant to pod shatter.

EXAMPLE 6 Production of Shatter-resistant B.napus Plants by AntisenseDownregulation of Multiple DZ-expressed Genes

[0114] In addition to DZ2 several other DZ-expressed genes have beenpreviously isolated and individually downregulated to result in B.napusplants that have increased resistance to pod shatter; namely Sac66 (WO96/30529 FIG. 15), DZ15 (FIG. 16) and OSR 7(9) (FIG. 17). It isanticipated that downregulation of more than one gene involved in podshatter will further increase resistance to pod shatter. This could beachieved by combining different transgenes by transformation withseveral transgenes each designed to downregulate a different DZ-expressed gene or by crossing together B.napus lines that individually aretransformed with such transgenes. Such methods are complex eitherinvolving transformation with a construct containing multiple chimericgenes or require the maintenance of several transgenic loci in thebreeding program. A preferred method is to transform with a chimericgene consisting of a single promoter driving expression of an antisenseor partial sense transcript which is comprised of elements of all theDZ-expressed genes to be downregulated. Similarly a single promotercould be used to drive the expression of multiple ribozymes eachtargeted against a different DZ-expressed gene. The use of a singlepromoter to drive expression of a combination of antisense, partialsense and ribozymes is also possible. Ideally the promoter will be podDZ-specific, however a useful promoter may be pod-specific or evenconstitutively active. A suitable DZ-specific promoter would be that ofDZ2. DZ2B, DZ2AT3 or ESJ2A.

[0115] Consequently the ESJ2A promoter was linked to a multipleantisense gene consisting of elements of Sac66, DZ2, DZ15 and OSR 7(9)in the following manner:- The original DZ15 PCR product in pCRII(Invitrogen) (see FIG. 16) was cloned as an EcoRI fragment intopBluescript SK (Stratagene) forming pDZ15-BS, such that the DZ15 3′ endis nearest the SstI site of the vector . T7 and DZ15RL primers were usedto PCR a 456 bp DZ15 fragment from pDZ15-BS which was cloned into theEcoRV site of pGEM5zf (Promega) forming pWP351, such that the DZ15 3′end is nearest the SphI site of the vector. 5′ AATACGACTCACTATAGG 3′ T75′ AACAGCTGAAAACCTCACGAAG 3′ DZ15RL

[0116] The EcoRI, NcoI fragment of pWP351 cloned between the EcoRI andNcoI sites of pDZ2-BS forming pWP356. pDZ2-BS consists of the DZ2 cDNAcloned as an EcoRI, XhoI fragment into pBluescript SK such that the 3′end is nearest the KpnI site of the vector. A 361 bp Sac66 fragment wasPCRed from the Sac66′cDNA (WO 96/30529) using the primers F1 and RIwhich introduce NcoI and PstI sites into the ends of the PCR product. 5′GGCCCATGGCTGCCAAGCTTTGAGTAGC 3′ F1 5′ GGCCTGCAGTGCCTAGGATCTGGAAGC 3′ RI

[0117] The Sac66 PCR product was cloned as an NcoI. EcoRI fragmentbetween the NcoI and EcoRI sites of pWP272 (WO 99/13089) formingpWP288A. EcoRI DZ15+DZ2 and OSR 7(9) fragments from pWP356 and pOSR7(9)-CRII were cloned into the EcoRI site of pWP288A such that DZ15+DZ2and OSR 7(9) are in an antisense orientation with respect to PGLpromoter. (POSR 7(9)-CRII consists of the 306 bp OSR 7(9) PCR fragment(see FIG. 17) cloned into pCRII (Invitrogen)). The chimericpPGL-antisense Sac66+DZ2+Dzl5 +OSR 7(9) gene was transferred as a XbaI.XhoI fragment into the XbaI and SalI sites of the binary vector pSCVnos-nptII (WO 95/20668) forming pWP357-SCV (FIG. 14). The pWP357-SCVbinary vector was transferred to the agrobacterial strain pGV2260 andtransformed B.napus plants produced by agrobacterial transformationessentially as described in Moloney M et al., (1989) Plant Cell Reports8, 238-242.

[0118] Resistance to podshatter was measured using an impact pendulumdevice (Liu X-Y, Macmillan RH and Burrow RP 1994 Journal of TextureStudies 25 p179-189) (Table 1). The mean energy values shown in Table 1represent the energy required to rupture the pod on impact with thependulum. These values are an average from measurements of 20 maturepods. The letters A to L indicate grouping of transformants withsignificantly different podshatter resistance (ie Group A issignificantly different from B when analysed by ANOVA using a FisherPLSD analysis with a significance level of 95% (Statview 512+). Lineswith a number of letters are not significantly different from otherlines sharing the same letter. The results shown in Table 1 indicatethat 24 lines exhibited significantly higher resistance to podshatterthan non-transformed controls whilst 17 lines were not significantlydifferent from the control. The degree and frequency of pod shatterresistance achieved with pWP357-SCV was greater than that obtained bytransformation with constructs that downregulate a single DZ-expressedgene. TABLE 1 PLANT ID MEAN ENERGY A213-24 6.752 A A213-53 5.203 BA213-34 3.864 C A213-21 3.673 C A213-4 3.54 C A213-61 3.516 C A214-303.46 C A214-27 3.397 C A213-8 3.277 C A213-70 3.271 C D A214-13 3.182 CD E A213-11 3.182 C D E F A213-69 3.005 D E F G A213-60 2.945 D E F G HA214-7 2.843 D E F G H I A213-64 2.687 D E F G H I J A214-14 2.613 D E FG H I J A213-28 2.581 D E F G H I J A213-9 2.547 E F G H I J A213-422.442 F G H I J A213-31 2.431 G H I J A214-10 2.42 H I J A213-38 2.295 HI J A214-8 2.26 H I J A213-19 2.213 H I J K A214-25 2.128 I J K A213-382.059 I J K A213-16 2.005 I J K A213-63 1.928 J K L A213-33 1.91 J K LA213-32 1.901 J K L A213-37 1.791 K L RV27CONT 1.787 K L A213-10 1.623 LA213-58 1.595 L A214-24 1.55 L A213-3 1.518 L A213-27 1.495 L A213-401.395 L A213-47 1.315 L A213-43 1.286 L A213-30 1.241 L

[0119] References

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[0121] 2. Christoffersen and Laties, Proc. Natl. Acad. Sci. 79, 40604063(1982)

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[0124] 5. Hocking P J and Mason L: Accumulation, distribution andredistribution of dry matter and mineral nutrients in fruits of canola(oilseed rape) and the effect of nitrogen fertiliser and windrowing.Australian Journal or Agriculture Research 44: 1377-1388 (1993)

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[0128] Plant Physiol 106: 601-606 (1994)

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[0131] 11. Meakin P J and Roberts J A: Dehiscence of fruit in oilseedrape (Brassica napus L.): anatomy of pod dehiscence. J Expt. Bot 41:995-1002 (1990a)

[0132] 12. Meakin P J and Roberts J A: Dehiscence of fruit in oilseedrape (Brassica napus L.): the role of cell wall degrading enzymes andethylene. J Expt. Bot 41: 1003-1011 (1990b)

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1 38 1 14 DNA Artificial Sequence Description of Artificial Sequenceoligo dT primer 7 1 tttttttttt ttgg 14 2 10 DNA Artificial SequenceDescription of Artificial Sequence Arbitrary primer A 2 agccagcgaa 10 319 DNA Artificial Sequence Description of Artificial Sequence PrimerDZ2BFL 3 aaccaagtca gtagaagtg 19 4 18 DNA Artificial SequenceDescription of Artificial Sequence Primer T7 4 aatacgactc actatagg 18 526 DNA Artificial Sequence Description of Artificial Sequence PrimerDZ2BGENF 5 ggctctagac gaactgcgga gcaagg 26 6 29 DNA Artificial SequenceDescription of Artificial Sequence Primer DZ2BGENR 6 ctgccatggtcggttttttt tcttcgaac 29 7 23 DNA Artificial Sequence Description ofArtificial Sequence Primer ATDZ2F 7 cactagtagg gcacgcgtgg tcg 23 8 27DNA Artificial Sequence Description of Artificial Sequence Primer ATDZ2R8 tccatggtcg atttcttttc tctcaag 27 9 28 DNA Artificial SequenceDescription of Artificial Sequence Primer DZ2FLA 9 ggcgaattcc ggtgaggaggcagtaatc 28 10 28 DNA Artificial Sequence Description of ArtificialSequence Primer DZ2RLA 10 ggcccatggc atacatacac acttagac 28 11 22 DNAArtificial Sequence Description of Artificial Sequence Primer DZ15RL 11aacagctgaa aacctcacga ag 22 12 28 DNA Artificial Sequence Description ofArtificial Sequence Primer F1 12 ggcccatggc tgccaagctt tgagtagc 28 13 27DNA Artificial Sequence Description of Artificial Sequence Primer R1 13ggcctgcagt gcctaggatc tggaagc 27 14 605 DNA Brassica napus CDS(20)..(427) 14 ggcacgagca gaatcgaag atg gca aca aaa tcc atg gga gat atcgag aaa 52 Met Ala Thr Lys Ser Met Gly Asp Ile Glu Lys 1 5 10 ata aagaag aaa cta aac gtg ttg atc gtc gat gat gat cca cta aac 100 Ile Lys LysLys Leu Asn Val Leu Ile Val Asp Asp Asp Pro Leu Asn 15 20 25 ctt ata attcat gag aag atc atc aaa gcg att ggg ggt att tca cag 148 Leu Ile Ile HisGlu Lys Ile Ile Lys Ala Ile Gly Gly Ile Ser Gln 30 35 40 aca gcg aat aacggt gag gag gca gta atc atc cac cgt gac ggc ggc 196 Thr Ala Asn Asn GlyGlu Glu Ala Val Ile Ile His Arg Asp Gly Gly 45 50 55 tca tct ttt gac cttatc cta atg gat aaa gaa atg ccc gag agg gat 244 Ser Ser Phe Asp Leu IleLeu Met Asp Lys Glu Met Pro Glu Arg Asp 60 65 70 75 ggt gtt tcg aca actaag aag cta aga gaa atg gaa gtg aag tca atg 292 Gly Val Ser Thr Thr LysLys Leu Arg Glu Met Glu Val Lys Ser Met 80 85 90 att gtt ggg gtg act tcactg gct gac aat gaa gag gag cgc agg gct 340 Ile Val Gly Val Thr Ser LeuAla Asp Asn Glu Glu Glu Arg Arg Ala 95 100 105 ttc atg gaa gct gga cttaac cat tgc ttg gca aaa ccg tta acc aag 388 Phe Met Glu Ala Gly Leu AsnHis Cys Leu Ala Lys Pro Leu Thr Lys 110 115 120 gac aag atc atc cct ctcatt aac caa ctc atg gat gct tgatggatat 437 Asp Lys Ile Ile Pro Leu IleAsn Gln Leu Met Asp Ala 125 130 135 atattttata ttatggaaac acacataataacgtctaagt gtgtatgtat gcatagatac 497 ttgcatgtgt gtgttttaga atttagggttctttatcgtc cgtgatatat aatcatgtaa 557 gttgttgctt taagcttata aaatatttaaataagggttt cctctacc 605 15 136 PRT Brassica napus 15 Met Ala Thr Lys SerMet Gly Asp Ile Glu Lys Ile Lys Lys Lys Leu 1 5 10 15 Asn Val Leu IleVal Asp Asp Asp Pro Leu Asn Leu Ile Ile His Glu 20 25 30 Lys Ile Ile LysAla Ile Gly Gly Ile Ser Gln Thr Ala Asn Asn Gly 35 40 45 Glu Glu Ala ValIle Ile His Arg Asp Gly Gly Ser Ser Phe Asp Leu 50 55 60 Ile Leu Met AspLys Glu Met Pro Glu Arg Asp Gly Val Ser Thr Thr 65 70 75 80 Lys Lys LeuArg Glu Met Glu Val Lys Ser Met Ile Val Gly Val Thr 85 90 95 Ser Leu AlaAsp Asn Glu Glu Glu Arg Arg Ala Phe Met Glu Ala Gly 100 105 110 Leu AsnHis Cys Leu Ala Lys Pro Leu Thr Lys Asp Lys Ile Ile Pro 115 120 125 LeuIle Asn Gln Leu Met Asp Ala 130 135 16 136 PRT Brassica napus 16 Met AlaThr Lys Ser Met Gly Asp Ile Glu Lys Ile Lys Lys Lys Leu 1 5 10 15 AsnVal Leu Ile Val Asp Asp Asp Pro Leu Asn Leu Ile Ile His Glu 20 25 30 LysIle Ile Lys Ala Ile Gly Gly Ile Ser Gln Thr Ala Asn Asn Gly 35 40 45 GluGlu Ala Val Ile Ile His Arg Asp Gly Gly Ser Ser Phe Asp Leu 50 55 60 IleLeu Met Asp Lys Glu Met Pro Glu Arg Asp Gly Val Ser Thr Thr 65 70 75 80Lys Lys Leu Arg Glu Met Glu Val Lys Ser Met Ile Val Gly Val Thr 85 90 95Ser Leu Ala Asp Asn Glu Glu Glu Arg Arg Ala Phe Met Glu Ala Gly 100 105110 Leu Asn His Cys Leu Ala Lys Pro Leu Thr Lys Asp Lys Ile Ile Pro 115120 125 Leu Ile Asn Gln Leu Met Asp Ala 130 135 17 132 PRT Escherichiacoli 17 Met Gln Glu Asn Tyr Lys Ile Leu Val Val Asp Asp Asp Met Arg Leu1 5 10 15 Arg Ala Leu Leu Glu Arg Tyr Leu Thr Glu Gln Gly Phe Gln ValArg 20 25 30 Ser Val Ala Asn Ala Glu Gln Met Asp Arg Leu Leu Thr Arg GluSer 35 40 45 Phe His Leu Met Val Leu Asp Leu Met Leu Pro Gly Glu Asp GlyLeu 50 55 60 Ser Ile Cys Arg Arg Leu Arg Ser Gln Ser Asn Pro Met Pro IleIle 65 70 75 80 Met Val Thr Ala Lys Gly Glu Glu Val Asp Arg Ile Val GlyLeu Glu 85 90 95 Ile Gly Ala Asp Asp Tyr Ile Pro Lys Pro Phe Asn Pro ArgGlu Leu 100 105 110 Leu Ala Arg Ile Arg Ala Val Leu Arg Arg Gln Ala AsnGlu Leu Pro 115 120 125 Gly Ala Pro Ser 130 18 126 PRT Escherichia coli18 Met Ala Arg Arg Ile Leu Val Val Glu Asp Glu Ala Pro Ile Arg Glu 1 510 15 Met Val Cys Phe Val Leu Glu Gln Asn Gly Phe Gln Pro Val Glu Ala 2025 30 Glu Asp Tyr Asp Ser Ala Val Asn Gln Leu Asn Glu Pro Trp Pro Asp 3540 45 Leu Ile Leu Leu Asp Trp Met Leu Pro Gly Gly Ser Gly Ile Gln Phe 5055 60 Ile Lys His Leu Lys Arg Glu Ser Met Thr Arg Asp Ile Pro Val Val 6570 75 80 Met Leu Thr Ala Arg Gly Glu Glu Glu Asp Arg Val Arg Gly Leu Glu85 90 95 Thr Gly Ala Asp Asp Tyr Ile Thr Lys Pro Phe Ser Pro Lys Glu Leu100 105 110 Val Ala Arg Ile Lys Ala Val Met Arg Arg Ile Ser Pro Met 115120 125 19 144 PRT Salmonella typhimurium 19 Met Gln Arg Gly Ile Val TrpVal Val Asp Asp Asp Ser Ser Ile Arg 1 5 10 15 Trp Val Leu Glu Arg AlaLeu Ala Gly Ala Gly Leu Thr Cys Thr Thr 20 25 30 Phe Glu Asn Gly Asn AsnThr Arg Cys Glu Val Leu Ala Ala Leu Ala 35 40 45 Ser Lys Thr Pro Asp ValLeu Leu Ser Asp Ile Arg Met Pro Gly Met 50 55 60 Asp Gly Leu Ala Leu LeuLys Gln Ile Lys Gln Arg His Pro Met Leu 65 70 75 80 Pro Val Ile Ile MetThr Ala Asn Thr Arg Cys His Ser Asp Leu Asp 85 90 95 Ala Ala Val Ser AlaTyr Gln Gln Gly Ala Phe Asp Tyr Leu Pro Lys 100 105 110 Pro Phe Asp IleAsp Glu Ala Val Ala Leu Val Glu Arg Ala Ile Ser 115 120 125 His Tyr GlnGlu Gln Gln Gln Pro Arg Asn Ile Glu Val Asn Gly Pro 130 135 140 20 124PRT Bacillus subtilis 20 Met Met Asn Glu Lys Ile Leu Ile Val Asp Asp GlnTyr Gly Ile Arg 1 5 10 15 Ile Leu Leu Asn Glu Val Phe Asn Lys Glu GlyTyr Gln Thr Phe Gln 20 25 30 Ala Ala Asn Gly Leu Gln Ala Leu Asp Ile ValThr Lys Glu Arg Pro 35 40 45 Asp Leu Val Leu Leu Asp Met Lys Ile Pro GlyMet Asp Gly Ile Glu 50 55 60 Ile Leu Lys Arg Met Lys Val Ile Asp Glu AsnIle Arg Val Ile Ile 65 70 75 80 Met Thr Ala Tyr Gly Glu Leu Asp Met IleGln Glu Ser Lys Glu Leu 85 90 95 Gly Ala Leu Thr His Phe Ala Lys Pro PheAsp Ile Asp Glu Ile Arg 100 105 110 Asp Ala Val Lys Lys Tyr Leu Pro LeuLys Ser Asn 115 120 21 129 PRT Escherichia coli 21 Met Ala Asp Lys GluLeu Lys Phe Leu Val Val Asp Asp Phe Ser Thr 1 5 10 15 Met Arg Arg IleVal Arg Asn Leu Leu Lys Glu Leu Gly Phe Asn Asn 20 25 30 Val Glu Glu AlaGlu Asp Gly Val Asp Ala Leu Asn Lys Leu Gln Ala 35 40 45 Gly Gly Tyr GlyPhe Val Ile Ser Asp Trp Asn Met Pro Asn Met Asp 50 55 60 Gly Leu Glu LeuLeu Lys Thr Ile Arg Ala Asp Gly Ala Met Ser Ala 65 70 75 80 Leu Pro ValLeu Met Val Thr Ala Glu Ala Lys Lys Glu Asn Ile Ile 85 90 95 Ala Ala AlaGln Ala Gly Ala Ser Gly Tyr Val Val Lys Pro Phe Thr 100 105 110 Pro AlaThr Leu Glu Glu Lys Leu Asn Lys Ile Phe Glu Lys Leu Gly 115 120 125 Met22 111 PRT Arabidopsis thaliana Unsure 67 Xaa= any amino acid 22 Leu LysVal Leu Val Met Asp Glu Asn Gly Val Ser Arg Met Val Thr 1 5 10 15 LysGly Leu Leu Val His Leu Gly Cys Glu Val Thr Thr Val Ser Ser 20 25 30 AsnGlu Glu Cys Leu Arg Val Val Ser His Glu His Lys Val Val Phe 35 40 45 MetAsp Val Cys Met Pro Gly Val Glu Asn Tyr Gln Ile Ala Leu Arg 50 55 60 IleHis Xaa Pro Leu Leu Val Ala Leu Ser Gly Asn Thr Asp Lys Ser 65 70 75 80Thr Lys Glu Lys Cys Met Ser Phe Gly Leu Asp Gly Val Leu Leu Lys 85 90 95Pro Val Ser Leu Asp Asn Ile Arg Asp Val Leu Ser Asp Leu Leu 100 105 11023 1716 DNA Brassica napus CDS (1516)..(1716) Unsure 48 n= anynucleotide 23 tatataaata cggtttaaca gatatgttct ggttataaat gtaattcnatgtgccnntca 60 anttttattt tnattngttn tactagggac attagtttta acnttttatatatcatgtaa 120 caaaaaaaaa aaaaacnttt tatatntcaa ctatgagcaa ttattcttatagtgttttct 180 ttttccagaa atttgacgac aacctaacta aaacaattta atttgacgttagttaagtaa 240 tttatataga tggataaatt gagcaagcac attacgaact gcggatcaaggagagtcaca 300 atttaattct tacgttatac acaaaattat ctaaatacta tatatatatacagctgcatg 360 ctacgataat gatcaaatgt ttatgtactt ttcagcgaaa attcttgtcgccatacatta 420 ctgtgttaat gaatcattaa atatgtgaag gaggaaaaga gtacaaaaggagttttgttg 480 aggcatttcg cagacactga aatgtgaata ataataaagg aattgccgaattgatttcta 540 gttggtgaag tgggtgaaaa ttgtatgtcc attgcttata aactataaaatataatatnt 600 tnatattatc actntggaca ttagtnngat agaccctagc taaaatttttaaaaattata 660 cattcatttt ctnaagtacc aaacttaatt atcacaatcg gataaaattgtttaagaaac 720 cattacaaac tcagcttgtg gactctgaga gaaactaaga gctagacatacggttagtag 780 tgtagccgca ttttttatgc ttaatttgct taagcatgac ttctatgctccttgatgata 840 tttattttaa tatcctagga catatggatt tgataaagat cttatcaacctttcaacaag 900 accattagct caacaaacaa aatactgaaa gtatataatc ttggttacagaattcttatg 960 ccaaaaatat cataatatat atagaattcg gttatgatta agatgaattatttaattaat 1020 atatttttca cttttgtttt cttatgtatt cttagtattt gttcaccatattgaccgatt 1080 ggtgtcatat tagtttggta agacaactca gttgcaacga tgcagattacatttcaggaa 1140 gattcatgta agaaagatat ttcgctttgt ggtgtgaaaa tatgcctctttcactttttt 1200 tcaactataa atttcgatcg atgtatctac gttcttaaca caattcacaatcttctttag 1260 aatccaaaat tgtaagccgc tttctaatct ctttctcagt atacatatgtaatatgtatg 1320 catatattat tattcataat acaaacacga acccatgcat gcaagaagatagttacacgc 1380 tcataacaaa cacaaaaaaa catacgcatg cattagaaca cttgtatgttaatttccata 1440 atgttttgca taaacattct tcgttttaat tagcttcttt ttgtgtgaagattgttcgaa 1500 gaaaaaaaac cgaag atg gca aca acg tca aca tcc acg gga gatatc aag 1551 Met Ala Thr Thr Ser Thr Ser Thr Gly Asp Ile Lys 1 5 10 aaaacc aag tca gta gaa gtg aag aag aaa ctt aac gtg ttg atc gtt 1599 Lys ThrLys Ser Val Glu Val Lys Lys Lys Leu Asn Val Leu Ile Val 15 20 25 gat gatgat aca gta att cgt aaa ctt cac gag aat atc atc aaa tcg 1647 Asp Asp AspThr Val Ile Arg Lys Leu His Glu Asn Ile Ile Lys Ser 30 35 40 atc ggt ggaatt tca cag acg gct aag aac ggt gag gag gca gtg aac 1695 Ile Gly Gly IleSer Gln Thr Ala Lys Asn Gly Glu Glu Ala Val Asn 45 50 55 60 atc cac cgcgac ggc aat gca 1716 Ile His Arg Asp Gly Asn Ala 65 24 67 PRT Brassicanapus 24 Met Ala Thr Thr Ser Thr Ser Thr Gly Asp Ile Lys Lys Thr Lys Ser1 5 10 15 Val Glu Val Lys Lys Lys Leu Asn Val Leu Ile Val Asp Asp AspThr 20 25 30 Val Ile Arg Lys Leu His Glu Asn Ile Ile Lys Ser Ile Gly GlyIle 35 40 45 Ser Gln Thr Ala Lys Asn Gly Glu Glu Ala Val Asn Ile His ArgAsp 50 55 60 Gly Asn Ala 65 25 576 DNA Brassica napus Unsure 6 n= anynucleotide 25 tcgtcnatga tgatcctgta atacgtaaac ttcacgagat tatcatcaaatcaatcggtg 60 gaatttcaca gacagctaag aacggtgagg aggcagtgaa catccaccgcgacggcaatg 120 catctttcga ccttatccta atggataaag aaatgcccga gagggatggactttcggcaa 180 ctaagaagct aagagaaatg aaagtgacgt ctatgattat tggggtgacgacactggctg 240 acaatgaaga ggaacgtaag gctttcatgg aagctggact taaccattgcttggcaaaac 300 ccttaagcaa agccaagatc ctccctctca tcaacaatct catggatgcttgatggatgg 360 atgaattgtc gccactacat atctacatta tacaaatatg aaaaacacatataataacgt 420 catacacctg tgtgtgtatg catagatatc tatccgcatg tgtgtttttagggttgttat 480 gtttgatttt tattgtgcgt ggcgtgatat acaatcangt nagtcgttacttttggctta 540 taaaataatg aataagattt gttaaaaata aaaaaa 576 26 116 PRTBrassica napus Unsure 2 Xaa= any amino acid 26 Val Xaa Asp Asp Pro ValIle Arg Lys Leu His Glu Ile Ile Ile Lys 1 5 10 15 Ser Ile Gly Gly IleSer Gln Thr Ala Lys Asn Gly Glu Glu Ala Val 20 25 30 Asn Ile His Arg AspGly Asn Ala Ser Phe Asp Leu Ile Leu Met Asp 35 40 45 Lys Glu Met Pro GluArg Asp Gly Leu Ser Ala Thr Lys Lys Leu Arg 50 55 60 Glu Met Lys Val ThrSer Met Ile Ile Gly Val Thr Thr Leu Ala Asp 65 70 75 80 Asn Glu Glu GluArg Lys Ala Phe Met Glu Ala Gly Leu Asn His Cys 85 90 95 Leu Ala Lys ProLeu Ser Lys Ala Lys Ile Leu Pro Leu Ile Asn Asn 100 105 110 Leu Met AspAla 115 27 818 DNA Arabidopsis thaliana CDS (180)..(605) Unsure 350 n=any nucleotide 27 atatatgtga tacagataca tctatataca aattaaacac gaaaccatacatgcacggtg 60 tgatcacaca cgcacacaca tagaaacata aacacgcaat aatttctatacagtttaatt 120 tcatttttaa cttacttctt tttttttggt gaagattctt gagagaaaagaaatcgaag 179 atg gca aca aaa tcc acc gga ggt acc gag aaa acc aag tcgata gaa 227 Met Ala Thr Lys Ser Thr Gly Gly Thr Glu Lys Thr Lys Ser IleGlu 1 5 10 15 gtg aag aag aaa cta atc aac gtg ttg atc gtc gat gat gatcca tta 275 Val Lys Lys Lys Leu Ile Asn Val Leu Ile Val Asp Asp Asp ProLeu 20 25 30 aac cgt aga ctc cac gag atg atc atc aaa acg atc gga gga atttct 323 Asn Arg Arg Leu His Glu Met Ile Ile Lys Thr Ile Gly Gly Ile Ser35 40 45 cag act gca aag aat ggc gaa gag gcn gtg atc ctc cac cgt gac ggc371 Gln Thr Ala Lys Asn Gly Glu Glu Xaa Val Ile Leu His Arg Asp Gly 5055 60 gaa gca tct ttc gac ctt att cta atg gat aag gaa atg cct gag agg419 Glu Ala Ser Phe Asp Leu Ile Leu Met Asp Lys Glu Met Pro Glu Arg 6570 75 80 gat gga gtt tcg aca att aag ang cta aga gaa atg aaa ggg acg tca467 Asp Gly Val Ser Thr Ile Lys Xaa Leu Arg Glu Met Lys Gly Thr Ser 8590 95 atg atc gtt ggg gta acg tca gta gct gac caa gaa gaa gag cgt aag515 Met Ile Val Gly Val Thr Ser Val Ala Asp Gln Glu Glu Glu Arg Lys 100105 110 gct ttt atg gaa gct ggg ctc aac cat tgc ttg gaa aaa ccc tta acc563 Ala Phe Met Glu Ala Gly Leu Asn His Cys Leu Glu Lys Pro Leu Thr 115120 125 aag gcc aag atc ttc ccg ctc att agc cac ctc ttc gat gct 605 LysAla Lys Ile Phe Pro Leu Ile Ser His Leu Phe Asp Ala 130 135 140tgatggatga aggctcatta atgtatctat attttcaatc atgaaatcac ctacacgtgt 665atttgacaca aaaatctgca tttgttgtga tatagggttt ctcatatcta tgtttgattt 725attttcttat cgtccgaggt aaaatcatgc aagtcatttc ttttggctaa taaaatatta 785aaataaggtt ttctcaaaaa aaaaaaaaaa aaa 818 28 142 PRT Arabidopsis thalianaUnsure 57 Xaa= any amino acid 28 Met Ala Thr Lys Ser Thr Gly Gly Thr GluLys Thr Lys Ser Ile Glu 1 5 10 15 Val Lys Lys Lys Leu Ile Asn Val LeuIle Val Asp Asp Asp Pro Leu 20 25 30 Asn Arg Arg Leu His Glu Met Ile IleLys Thr Ile Gly Gly Ile Ser 35 40 45 Gln Thr Ala Lys Asn Gly Glu Glu XaaVal Ile Leu His Arg Asp Gly 50 55 60 Glu Ala Ser Phe Asp Leu Ile Leu MetAsp Lys Glu Met Pro Glu Arg 65 70 75 80 Asp Gly Val Ser Thr Ile Lys XaaLeu Arg Glu Met Lys Gly Thr Ser 85 90 95 Met Ile Val Gly Val Thr Ser ValAla Asp Gln Glu Glu Glu Arg Lys 100 105 110 Ala Phe Met Glu Ala Gly LeuAsn His Cys Leu Glu Lys Pro Leu Thr 115 120 125 Lys Ala Lys Ile Phe ProLeu Ile Ser His Leu Phe Asp Ala 130 135 140 29 1324 DNA Arabidopsisthaliana CDS (1200)..(1322) Unsure 1142 n= any nucleotide 29 gtaatgcgactcactatagg gcacgcgtgg tcgacggccc gggctggtcc tcattcgtat 60 tgggcccaatgggctactaa aacagtttca cgattgtttt tttttttttt tttttaattt 120 ttaacatgtatgtgggatat ttggctataa attatgtaaa aaatttcacg atagattgtt 180 gaatttttgaatttcgagtt aaaatatctt caaattacct cacatttaca aaaaggtaga 240 actgttgaaaaactaatgct ctataaaaca ctagacaata acaaaatacg taatgcgtaa 300 agaacctaaattatgatttt atttatcttt cttccttttt ccgtgagtat aagccatttt 360 tcatagtaaagcattacgaa tacgacattg aacactactg acatataaag tagtagattt 420 tgatgggttaacttgtatgc ttaatttgct taagcatgaa cttcaatgct tttataaaag 480 tacttcatgagaatattcct cgttctatac tagcagaagg gttcgatagt gattttacaa 540 ccgttcaacaaaacctttaa acccaaaaaa ccaaagaatg aaagtatcta aacttgatta 600 tacatttcttgtctaaatta tcaaataaca tactctcttt tgtttactta taaacgatat 660 gaaagaaataaataaaaaga acatagaatc ttattatgat ctagaagaat taattaaaga 720 aatatatatatatttttttt catttctact catgtttctt atacattctt taaatttgtt 780 caccattttgatttacttgt tctcatatta gtttgttata caactcactt agaataatgt 840 agattacatttcagccaaat tcatgtaaaa gatgcttttc tttgtgatgt ttttaaaatg 900 ctttcttttcactttttttc tttcttaact ataaatcttg atcgaatgcc taccttctta 960 gaacataagatcttctttaa aatccaaaat cgtaggccac tatttcatta tacttatgta 1020 atatatgtgatacagataca tntatataca aattaaacac gaaaccatac atgcacggtg 1080 tgatcacacacgcacacaca tagaaacata aacacgcaat aatttctata cagtttaatt 1140 tcatttttaacttacttctt tttttttggt gaagattctt gagagaaaag aaatcgaag 1199 atg gca acaaaa tcc acc gga ggt acc gag aaa acc aag tcg ata gaa 1247 Met Ala Thr LysSer Thr Gly Gly Thr Glu Lys Thr Lys Ser Ile Glu 1 5 10 15 gtg aag aagaaa cta atc aac gtg ttg atc gtc gat gat gat cca tta 1295 Val Lys Lys LysLeu Ile Asn Val Leu Ile Val Asp Asp Asp Pro Leu 20 25 30 aac cgt aga ctccac gag tgt cat caa aa 1324 Asn Arg Arg Leu His Glu Cys His Gln 35 40 3041 PRT Arabidopsis thaliana 30 Met Ala Thr Lys Ser Thr Gly Gly Thr GluLys Thr Lys Ser Ile Glu 1 5 10 15 Val Lys Lys Lys Leu Ile Asn Val LeuIle Val Asp Asp Asp Pro Leu 20 25 30 Asn Arg Arg Leu His Glu Cys His Gln35 40 31 1657 DNA Brassica napus CDS (145)..(1443) 31 ggcatcacgagggtacccgt aaatcccacc atacaacaaa gttctgtgaa agtctcccaa 60 aaactgcaaagagtctcata ttagttctta ctctcagaaa taaaacacac tctttctgaa 120 aagattagcgtttcaaaccc cgaa atg gcc cgt tgt cat gga agt ctt gct 171 Met Ala Arg CysHis Gly Ser Leu Ala 1 5 att ttc tta tgc gtt ctt ttg atg ctc gct tgc tgccaa gct ttg agt 219 Ile Phe Leu Cys Val Leu Leu Met Leu Ala Cys Cys GlnAla Leu Ser 10 15 20 25 agc aac gta gat gat gga tat ggt cat gaa gat ggaagc ttc gaa acc 267 Ser Asn Val Asp Asp Gly Tyr Gly His Glu Asp Gly SerPhe Glu Thr 30 35 40 gat agt tta atc aag ctc aac aac gac gac gac gtt cttacc ttg aaa 315 Asp Ser Leu Ile Lys Leu Asn Asn Asp Asp Asp Val Leu ThrLeu Lys 45 50 55 agc tcc gat aga ccc act acc gaa tca tca act gtt agt gtttcg aac 363 Ser Ser Asp Arg Pro Thr Thr Glu Ser Ser Thr Val Ser Val SerAsn 60 65 70 ttc gga gca aaa ggt gat gga aaa acc gat gat act cag gct ttcaag 411 Phe Gly Ala Lys Gly Asp Gly Lys Thr Asp Asp Thr Gln Ala Phe Lys75 80 85 aaa gca tgg aag aag gca tgt tca aca aat gga gtg act act ttc ttg459 Lys Ala Trp Lys Lys Ala Cys Ser Thr Asn Gly Val Thr Thr Phe Leu 9095 100 105 att cct aaa ggg aag act tat ctc ctt aag tct att aga ttc agaggc 507 Ile Pro Lys Gly Lys Thr Tyr Leu Leu Lys Ser Ile Arg Phe Arg Gly110 115 120 cca tgc aaa tca tta cgt agc ttc cag atc cta ggc act tta tcagct 555 Pro Cys Lys Ser Leu Arg Ser Phe Gln Ile Leu Gly Thr Leu Ser Ala125 130 135 tct aca aaa cga tcg gat tac agt aat gac aag aac cac tgg cttatt 603 Ser Thr Lys Arg Ser Asp Tyr Ser Asn Asp Lys Asn His Trp Leu Ile140 145 150 ttg gag gac gtt aat aat cta tca atc gat ggc ggc tcg gcg gggatt 651 Leu Glu Asp Val Asn Asn Leu Ser Ile Asp Gly Gly Ser Ala Gly Ile155 160 165 gtt gat ggc aac gga aaa atc tgg tgg caa aac tca tgc aaa atcgac 699 Val Asp Gly Asn Gly Lys Ile Trp Trp Gln Asn Ser Cys Lys Ile Asp170 175 180 185 aaa tct aag cca tgc aca aaa gcg cca acg gct ctt act ctctac aac 747 Lys Ser Lys Pro Cys Thr Lys Ala Pro Thr Ala Leu Thr Leu TyrAsn 190 195 200 cta aac aat ttg aat gtg aag aat ctg aga gtg aga aat gcacag cag 795 Leu Asn Asn Leu Asn Val Lys Asn Leu Arg Val Arg Asn Ala GlnGln 205 210 215 att cag att tcg att gag aaa tgc aac agt gtt gat gtt aagaat gtt 843 Ile Gln Ile Ser Ile Glu Lys Cys Asn Ser Val Asp Val Lys AsnVal 220 225 230 aag atc act gct cct ggc gat agt ccc aac acg gat ggt attcat atc 891 Lys Ile Thr Ala Pro Gly Asp Ser Pro Asn Thr Asp Gly Ile HisIle 235 240 245 gtt gct act aaa aac att cga atc tcc aat tca gac att gggaca ggt 939 Val Ala Thr Lys Asn Ile Arg Ile Ser Asn Ser Asp Ile Gly ThrGly 250 255 260 265 gat gat tgc ata tcc att gag gat gga tcg caa aat gttcaa atc aat 987 Asp Asp Cys Ile Ser Ile Glu Asp Gly Ser Gln Asn Val GlnIle Asn 270 275 280 gat tta act tgc ggc ccc ggt cat ggc atc agc att ggaagc ttg ggg 1035 Asp Leu Thr Cys Gly Pro Gly His Gly Ile Ser Ile Gly SerLeu Gly 285 290 295 gat gac aat tcc aaa gct tat gta tcg gga att aat gtggat ggt gct 1083 Asp Asp Asn Ser Lys Ala Tyr Val Ser Gly Ile Asn Val AspGly Ala 300 305 310 acg ctc tct gag act gac aat gga gta aga atc aag acttac cag gga 1131 Thr Leu Ser Glu Thr Asp Asn Gly Val Arg Ile Lys Thr TyrGln Gly 315 320 325 ggg tca gga act gct aag aac att aaa ttc caa aac attcgt atg gat 1179 Gly Ser Gly Thr Ala Lys Asn Ile Lys Phe Gln Asn Ile ArgMet Asp 330 335 340 345 aat gtc aag aat ccg atc ata atc gac cag aac tactgc gac aag gac 1227 Asn Val Lys Asn Pro Ile Ile Ile Asp Gln Asn Tyr CysAsp Lys Asp 350 355 360 aaa tgc gaa caa caa gaa tct gcg gtt caa gtg aacaat gtc gtg tat 1275 Lys Cys Glu Gln Gln Glu Ser Ala Val Gln Val Asn AsnVal Val Tyr 365 370 375 cgg aac ata caa ggt acg agc gca acg gat gtg gcgata atg ttt aat 1323 Arg Asn Ile Gln Gly Thr Ser Ala Thr Asp Val Ala IleMet Phe Asn 380 385 390 tgc agt gtg aaa tat cca tgc caa ggt att gtg cttgag aat gtg aac 1371 Cys Ser Val Lys Tyr Pro Cys Gln Gly Ile Val Leu GluAsn Val Asn 395 400 405 atc aaa gga gga aaa gct tct tgc aaa aat gtc aatgtt aag gat aaa 1419 Ile Lys Gly Gly Lys Ala Ser Cys Lys Asn Val Asn ValLys Asp Lys 410 415 420 425 ggc acc gtt tct cct aaa tgc cct taattactaagttgattatg taatatacat 1473 Gly Thr Val Ser Pro Lys Cys Pro 430aaatacgtat tatatgtggt tatagatgcc atctatatcc ttatctacgt attgattctc 1533gatatatata gaaaactaag gatttatggg aatatacata caatagttga gataattgtt 1593gtcttgtata tggttcactg aagttgattg cttgtccacg aataaatgaa taatgtcatt 1653tgtc 1657 32 433 PRT Brassica napus 32 Met Ala Arg Cys His Gly Ser LeuAla Ile Phe Leu Cys Val Leu Leu 1 5 10 15 Met Leu Ala Cys Cys Gln AlaLeu Ser Ser Asn Val Asp Asp Gly Tyr 20 25 30 Gly His Glu Asp Gly Ser PheGlu Thr Asp Ser Leu Ile Lys Leu Asn 35 40 45 Asn Asp Asp Asp Val Leu ThrLeu Lys Ser Ser Asp Arg Pro Thr Thr 50 55 60 Glu Ser Ser Thr Val Ser ValSer Asn Phe Gly Ala Lys Gly Asp Gly 65 70 75 80 Lys Thr Asp Asp Thr GlnAla Phe Lys Lys Ala Trp Lys Lys Ala Cys 85 90 95 Ser Thr Asn Gly Val ThrThr Phe Leu Ile Pro Lys Gly Lys Thr Tyr 100 105 110 Leu Leu Lys Ser IleArg Phe Arg Gly Pro Cys Lys Ser Leu Arg Ser 115 120 125 Phe Gln Ile LeuGly Thr Leu Ser Ala Ser Thr Lys Arg Ser Asp Tyr 130 135 140 Ser Asn AspLys Asn His Trp Leu Ile Leu Glu Asp Val Asn Asn Leu 145 150 155 160 SerIle Asp Gly Gly Ser Ala Gly Ile Val Asp Gly Asn Gly Lys Ile 165 170 175Trp Trp Gln Asn Ser Cys Lys Ile Asp Lys Ser Lys Pro Cys Thr Lys 180 185190 Ala Pro Thr Ala Leu Thr Leu Tyr Asn Leu Asn Asn Leu Asn Val Lys 195200 205 Asn Leu Arg Val Arg Asn Ala Gln Gln Ile Gln Ile Ser Ile Glu Lys210 215 220 Cys Asn Ser Val Asp Val Lys Asn Val Lys Ile Thr Ala Pro GlyAsp 225 230 235 240 Ser Pro Asn Thr Asp Gly Ile His Ile Val Ala Thr LysAsn Ile Arg 245 250 255 Ile Ser Asn Ser Asp Ile Gly Thr Gly Asp Asp CysIle Ser Ile Glu 260 265 270 Asp Gly Ser Gln Asn Val Gln Ile Asn Asp LeuThr Cys Gly Pro Gly 275 280 285 His Gly Ile Ser Ile Gly Ser Leu Gly AspAsp Asn Ser Lys Ala Tyr 290 295 300 Val Ser Gly Ile Asn Val Asp Gly AlaThr Leu Ser Glu Thr Asp Asn 305 310 315 320 Gly Val Arg Ile Lys Thr TyrGln Gly Gly Ser Gly Thr Ala Lys Asn 325 330 335 Ile Lys Phe Gln Asn IleArg Met Asp Asn Val Lys Asn Pro Ile Ile 340 345 350 Ile Asp Gln Asn TyrCys Asp Lys Asp Lys Cys Glu Gln Gln Glu Ser 355 360 365 Ala Val Gln ValAsn Asn Val Val Tyr Arg Asn Ile Gln Gly Thr Ser 370 375 380 Ala Thr AspVal Ala Ile Met Phe Asn Cys Ser Val Lys Tyr Pro Cys 385 390 395 400 GlnGly Ile Val Leu Glu Asn Val Asn Ile Lys Gly Gly Lys Ala Ser 405 410 415Cys Lys Asn Val Asn Val Lys Asp Lys Gly Thr Val Ser Pro Lys Cys 420 425430 Pro 33 569 DNA Brassica napus CDS (3)..(311) 33 ag gtg acc gtt gctgat ggc aat gtg ctg gtc aag cga gag gta gac 47 Val Thr Val Ala Asp GlyAsn Val Leu Val Lys Arg Glu Val Asp 1 5 10 15 ggt ggc ttg gag aca gttaaa gtc aaa ttg cca gct gtc att agc gcc 95 Gly Gly Leu Glu Thr Val LysVal Lys Leu Pro Ala Val Ile Ser Ala 20 25 30 gac ttg cgg ctc aat gag ccgcgg tac gct act ctg ccc aat atc atg 143 Asp Leu Arg Leu Asn Glu Pro ArgTyr Ala Thr Leu Pro Asn Ile Met 35 40 45 aag gcc aag aag aag ccc atc aaaaag ctc aca gcc aca gat gtc ggt 191 Lys Ala Lys Lys Lys Pro Ile Lys LysLeu Thr Ala Thr Asp Val Gly 50 55 60 gtg gac ttg gcg cca cgt caa caa gtgttg agc gta gaa gac ccg ccc 239 Val Asp Leu Ala Pro Arg Gln Gln Val LeuSer Val Glu Asp Pro Pro 65 70 75 acc aga cag gct ggt tcc att gtg cct gatgtc gac act ctc atc acc 287 Thr Arg Gln Ala Gly Ser Ile Val Pro Asp ValAsp Thr Leu Ile Thr 80 85 90 95 aag ttg aaa gaa aag ggt cat ttgtaatgcaatg tcaccaatac agttgtttta 341 Lys Leu Lys Glu Lys Gly His Leu 100gttcttacaa attcttcgtg aggttttcag ctgttaccaa taatattttt tcaaaatcga 401ttttatttta cttgtaattt aaaagatcaa atattaatac aatgaacatt tttgtaacag 461caatcttttt tttatatttt ggagatttca tcgacttatg tcataattat ttttatcaat 521ttattgttgt ttgttagtga tataataaag tatattttct ggtcaaaa 569 34 103 PRTBrassica napus 34 Val Thr Val Ala Asp Gly Asn Val Leu Val Lys Arg GluVal Asp Gly 1 5 10 15 Gly Leu Glu Thr Val Lys Val Lys Leu Pro Ala ValIle Ser Ala Asp 20 25 30 Leu Arg Leu Asn Glu Pro Arg Tyr Ala Thr Leu ProAsn Ile Met Lys 35 40 45 Ala Lys Lys Lys Pro Ile Lys Lys Leu Thr Ala ThrAsp Val Gly Val 50 55 60 Asp Leu Ala Pro Arg Gln Gln Val Leu Ser Val GluAsp Pro Pro Thr 65 70 75 80 Arg Gln Ala Gly Ser Ile Val Pro Asp Val AspThr Leu Ile Thr Lys 85 90 95 Leu Lys Glu Lys Gly His Leu 100 35 306 DNABrassica napus CDS (3)..(305) 35 gg ttg ggt cga acc ata ggt gga aag cttctt tct ctc tcg ctt gac 47 Leu Gly Arg Thr Ile Gly Gly Lys Leu Leu SerLeu Ser Leu Asp 1 5 10 15 aaa tcc tct ggt tcg ggt ttt cag tcc cat caggag ttt ctc tat ggt 95 Lys Ser Ser Gly Ser Gly Phe Gln Ser His Gln GluPhe Leu Tyr Gly 20 25 30 aaa gct gag gtt caa atg aaa ctt gtc cct ggt aactct gct gga aca 143 Lys Ala Glu Val Gln Met Lys Leu Val Pro Gly Asn SerAla Gly Thr 35 40 45 gtc aca aca ttc tat ctt aaa tca ccg gga act aca tgggat gag atc 191 Val Thr Thr Phe Tyr Leu Lys Ser Pro Gly Thr Thr Trp AspGlu Ile 50 55 60 gat ttc gag ttc ttg gga aac ata agt ggc cat ccc tat actctc cat 239 Asp Phe Glu Phe Leu Gly Asn Ile Ser Gly His Pro Tyr Thr LeuHis 65 70 75 act aat gtt tac aca cga agg ctc tgg aga caa aga aca gca gtttca 287 Thr Asn Val Tyr Thr Arg Arg Leu Trp Arg Gln Arg Thr Ala Val Ser80 85 90 95 tct atg gtt cga ccc gac c 306 Ser Met Val Arg Pro Asp 100 36101 PRT Brassica napus 36 Leu Gly Arg Thr Ile Gly Gly Lys Leu Leu SerLeu Ser Leu Asp Lys 1 5 10 15 Ser Ser Gly Ser Gly Phe Gln Ser His GlnGlu Phe Leu Tyr Gly Lys 20 25 30 Ala Glu Val Gln Met Lys Leu Val Pro GlyAsn Ser Ala Gly Thr Val 35 40 45 Thr Thr Phe Tyr Leu Lys Ser Pro Gly ThrThr Trp Asp Glu Ile Asp 50 55 60 Phe Glu Phe Leu Gly Asn Ile Ser Gly HisPro Tyr Thr Leu His Thr 65 70 75 80 Asn Val Tyr Thr Arg Arg Leu Trp ArgGln Arg Thr Ala Val Ser Ser 85 90 95 Met Val Arg Pro Asp 100 37 27 DNAArtificial Sequence GW1 37 tgattaatgc ctcctctccg ttattcg 27 38 27 DNAArtificial Sequence AT3GW2 38 ttgcagtctg agaaattcct ccgatcg 27

1. Nucleic acid encoding a signal transduction protein involved in theprocess of dehiscence.
 2. Nucleic acid as claimed in claim 1 wherein theprocess involves the production of a hydrolytic enzyme.
 3. Nucleic acidas claimed in claim 1 or claim 2 which is naturally expressed in adehiscence zone.
 4. Nucleic acid encoding a protein wherein the protein:a) comprises the amino acid sequence shown in FIG. 1 or; b) has one ormore amino acid deletions, insertions or substitutions relative to aprotein as defined in a) above and has at least 40% amino acid sequenceidentity therewith; or c) is a fragment of a protein as defined in a) orb) above, which is at least 10 amino acids long.
 5. Nucleic acid asclaimed in any one of claims 1 to 4 which comprises the sequence set outin FIG. 1 or a fragment thereof which is at least 30 bases long. 6.Nucleic acid, as claimed in any one of claims 1 to 5 in combination withone or more further nucleic acid sequence which is dehiscence-zoneexpressed.
 7. Nucleic acid which is antisense to nucleic acid as claimedin any one of claims 1 to
 6. 8. Nucleic acid as claimed in any one ofclaims 1 to 7 including a promoter or other regulatory sequence whichcontrols expression of the nucleic acid.
 9. Nucleic acid which is thenaturally occurring promoter or other regulatory sequence which controlsexpression of nucleic acid as claimed in any one of claims 1 to
 8. 10.Nucleic acid as claimed in any one of claims 1 to 9 which is in the formof a vector.
 11. A cell comprising nucleic acid as claimed in any one ofclaims 1 to
 10. 12. A plant cell as claimed in claim
 11. 13. A processfor obtaining a cell as claimed in claim 11 or claim 12 comprisingintroducing nucleic acid as claimed in any one of claims 1 to 10 intosaid cell.
 14. A plant or a part thereof comprising a cell as claimed inclaim 11 or claim
 12. 15. Propagating material or a seed comprising acell as claimed in claim 11 or claim
 12. 16. A process for obtaining aplant or plant part as claimed in claim 14 or claim 15 comprisingobtaining a cell as claimed in claim 11 and growth thereof or obtaininga plant, plant part, or propagating material as claimed in claim 14 orclaim 15 and growth thereof.
 17. A signal transduction protein involvedin the process of plant dehiscence.
 18. A protein which: a) comprisesthe amino acid sequence shown in FIG. 1 or; b) has one or more aminoacid deletions, insertions or substitutions relative to a protein asdefined in a) above, and has at least 40% amino acid sequence identitytherewith; or c) a fragment of a protein as defined in a) or b) abovewhich is at least 10 amino acids long.
 19. A protein as claimed in claim17 or claim 18 which is isolated or recombinant.
 20. A process forregulating/controlling dehiscence in a plant or a part thereof, theprocess comprising obtaining a plant or part thereof as claimed in claim14.
 21. A process as claimed in claim 20 which comprises obtaining aplant cell as claimed in claim 21 or part of a plant as claimed in claim14 and deriving a plant therefrom.
 22. A process as claimed in claim 20which comprises obtaining propagating material or a seed as claimed inclaim 15 and deriving a plant therefrom
 23. A process as claimed inclaim 20 wherein the dehiscence is of a pod or of an anther.
 24. Use ofnucleic acid as claimed in any one of claims 1 to 10 in theregulation/control of plant dehiscence.
 25. Use of nucleic acid asclaimed in any one of claims 1 to 10 as a probe.
 26. Use of nucleic acidas claimed in any one of claims 1 to 10 in the production of a cell,tissue, plant part thereof or propagating material.
 27. Nucleic acidcomprising one or more of the underlined sequences as set out in FIG. 1,or one or more of the primer sequences in FIG. 5, 9 and/or
 11. 28. Useof the nucleic acid as claimed in claim 27 as a PCR primer.
 29. Use of aprotein as claimed in any one of claims 17 to 19 as a probe.